Figure 10: A simplified schematic depiction of the role of uPAR in angiogenesis: The uPAR/Pro-uPA interaction leads to the generation of active uPA on the cell surface. This complex binds to vitronectin in the extracellular matrix, allowing interaction with its transmembrane partners, the α/β integrins. This leads to a cascade of activation events resulting in tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin molecules. Through recruitment of other adaptor molecules, such as Src and p130Cas-CRK complex (not shown), Rac is activated. Activated Rac then induces actin filament assembly leading to membrane protrusion and motility. Formation of focal adhesion complexes enables cellular adhesion and migration. The uPA/uPAR complex also generates the serine protease, plasmin that degrades the extracellular matrix (ECM) thereby stimulating conditions for migration and proliferation. The transmembrane partnership between uPA/uPAR and α/β integrins also activates mitogen activated kinase signaling molecules, MEK and ERK1/2, as well as the phosphoinositide 3-kinase (PI3K)/Akt signaling axis. Thus uPAR-dependent multiple signaling events regulates cellular adhesion, proliferation, and migration, events associated with angiogenesis.

Mentions:
The uPAR protein is a versatile molecule that coordinates a number of cellular processes [9,58]. These include its interaction with Vn [8], which is required for maintaining normal physiological activities, such as haemostasis [59] and angiogenesis [60] (Figure 10). Vn levels are known to increase during pathological conditions of rheumatoid arthritis [61] and tumor invasion [62]. The role of uPAR in pathological angiogenesis is well established and down-regulating uPAR expression in cancerous cells attenuates tumor growth, accompanied by decreased cellular migration, invasion, and adhesion [63-65]. In this study, uPAR-/- ECs demonstrated enhanced adhesion compared to WT cells when tested on Vn and collagen, but not on fibronectin. It is known that uPAR is required for cells to adhere to Vn and collagen [27,28], which then regulates integrin-mediated signaling [66]. In the absence of uPAR the ECs show a "fried egg" morphology and lack lamellipodia formation when plated on Vn compared to WT cells. However these cells exhibited normal cell morphology on collagen. Thus, further evaluations of different focal adhesion and signaling molecules were performed on ECs adherent on Vn. It was also observed that levels of β1-integrins in uPAR-/- ECs were increased after adhesion on Vn with a concomitant increase in focal adhesion protein FAK(P-Tyr925). Thus in the absence of uPAR, which would otherwise bind to Vn, the presence of enhanced levels of β1-integrin and FAK(P-Tyr925) in uPAR-/- ECs is most likely responsible for enhanced adhesion of these cells to Vn. Indeed, it has been reported that HEK293 cells not expressing uPAR are capable of integrin-mediated adhesion to Vn but fail to do so under integrin blocking conditions [21]. Aberrant integrin and FAK signaling is known to facilitate tumor cell invasion and metastasis. However, its activation in each cell type is interpreted differently and current knowledge regarding integrin/FAK signaling is contradictory and dependent on interactions with other focal adhesion proteins, such as paxillin, p130Cas, GRb2, and Shc [67,68]. As shown schematically in Figure 10, interaction of uPA with uPAR results in generation of plasmin that degrades the extracellular matrix facilitating cellular migration and proliferation. Even though the uPAR-/- ECs exhibited increased adhesion on Vn and collagen, a total uPAR deficiency would lead to compromised uPA/uPAR interaction, thus preventing uPA/uPAR-dependent proteolysis that would otherwise positively influence cellular migration and proliferation.

Figure 10: A simplified schematic depiction of the role of uPAR in angiogenesis: The uPAR/Pro-uPA interaction leads to the generation of active uPA on the cell surface. This complex binds to vitronectin in the extracellular matrix, allowing interaction with its transmembrane partners, the α/β integrins. This leads to a cascade of activation events resulting in tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin molecules. Through recruitment of other adaptor molecules, such as Src and p130Cas-CRK complex (not shown), Rac is activated. Activated Rac then induces actin filament assembly leading to membrane protrusion and motility. Formation of focal adhesion complexes enables cellular adhesion and migration. The uPA/uPAR complex also generates the serine protease, plasmin that degrades the extracellular matrix (ECM) thereby stimulating conditions for migration and proliferation. The transmembrane partnership between uPA/uPAR and α/β integrins also activates mitogen activated kinase signaling molecules, MEK and ERK1/2, as well as the phosphoinositide 3-kinase (PI3K)/Akt signaling axis. Thus uPAR-dependent multiple signaling events regulates cellular adhesion, proliferation, and migration, events associated with angiogenesis.

Mentions:
The uPAR protein is a versatile molecule that coordinates a number of cellular processes [9,58]. These include its interaction with Vn [8], which is required for maintaining normal physiological activities, such as haemostasis [59] and angiogenesis [60] (Figure 10). Vn levels are known to increase during pathological conditions of rheumatoid arthritis [61] and tumor invasion [62]. The role of uPAR in pathological angiogenesis is well established and down-regulating uPAR expression in cancerous cells attenuates tumor growth, accompanied by decreased cellular migration, invasion, and adhesion [63-65]. In this study, uPAR-/- ECs demonstrated enhanced adhesion compared to WT cells when tested on Vn and collagen, but not on fibronectin. It is known that uPAR is required for cells to adhere to Vn and collagen [27,28], which then regulates integrin-mediated signaling [66]. In the absence of uPAR the ECs show a "fried egg" morphology and lack lamellipodia formation when plated on Vn compared to WT cells. However these cells exhibited normal cell morphology on collagen. Thus, further evaluations of different focal adhesion and signaling molecules were performed on ECs adherent on Vn. It was also observed that levels of β1-integrins in uPAR-/- ECs were increased after adhesion on Vn with a concomitant increase in focal adhesion protein FAK(P-Tyr925). Thus in the absence of uPAR, which would otherwise bind to Vn, the presence of enhanced levels of β1-integrin and FAK(P-Tyr925) in uPAR-/- ECs is most likely responsible for enhanced adhesion of these cells to Vn. Indeed, it has been reported that HEK293 cells not expressing uPAR are capable of integrin-mediated adhesion to Vn but fail to do so under integrin blocking conditions [21]. Aberrant integrin and FAK signaling is known to facilitate tumor cell invasion and metastasis. However, its activation in each cell type is interpreted differently and current knowledge regarding integrin/FAK signaling is contradictory and dependent on interactions with other focal adhesion proteins, such as paxillin, p130Cas, GRb2, and Shc [67,68]. As shown schematically in Figure 10, interaction of uPA with uPAR results in generation of plasmin that degrades the extracellular matrix facilitating cellular migration and proliferation. Even though the uPAR-/- ECs exhibited increased adhesion on Vn and collagen, a total uPAR deficiency would lead to compromised uPA/uPAR interaction, thus preventing uPA/uPAR-dependent proteolysis that would otherwise positively influence cellular migration and proliferation.

Bottom Line:
This study focuses on the effect of uPAR deficiency (uPAR-/-) on angiogenic function and associated cytoskeletal organization.VEGF-enriched Matrigel implants from uPAR-/- mice demonstrated a lack of mature vessel formation compared to WT mice.Collectively, these results indicate that a uPAR deficiency leads to decreased angiogenic functions of endothelial cells.